Microfibril-associated glycoproteins attenuating pulmonary fibrosis

项目概要/摘要 特发性肺纤维化（IPF）是一种进行性肺部疾病，治疗选择有限且尚不清楚 发病机制。增殖的成纤维细胞群导致细胞外基质 (ECM) 沉积，驱动 由细胞因子转化生长介导的进行性气流受限和 IPF 缺氧特征 β因子（TGFB）。直接抑制 TGFB 会导致无法忍受的副作用，因此人们越来越关注 相反，调节其调节的疗法。微原纤维相关糖蛋白 1 和 2（Magp1 和 Magp2， 或 Magps）是成纤维细胞产生的蛋白质，分泌至 ECM、锚定至微纤维并结合 TGFB 在基础条件下限制其信号传导，但在纤维化中的作用尚不清楚。 Magp 敲除 (KO) 小鼠 在各种组织中表现出 TGFB 信号传导增强，并因骨髓而导致免疫细胞发生改变 TGFB 的作用。在单细胞 RNASeq 筛选中，编码这些蛋白质的基因显着上调 在纤维化肺的成纤维细胞中，Magp1 和 2 的联合敲除导致博来霉素后更严重的纤维化 小鼠受伤。这表明 Magps 作为纤维化信号传导的抑制剂，并对 Magp1 进行后续研究 两只 KO 小鼠表明，两种蛋白质的缺乏是产生促纤维化表型所必需的。 哪些成纤维细胞群产生 Magps 以及它们如何限制纤维化尚不清楚。我们假设 Magp 表达是对肺损伤的适应性反应，并通过隔离 ECM 中的 TGFB 来限制纤维化， 将通过三个具体目标探索这一中心假设：1）评估 Magp1 和 2 在肺纤维化中的作用。 2) 评估 Magp 对 TGFB 信号传导和免疫的影响 肺纤维化的反应。 3) 表征产生 Magp 的成纤维细胞群体。使用组合 KO 和诱导型/组织特异性 KO 小鼠，我们将确定这些蛋白质与 纤维化以及它们在体内是否表现出多余的抗纤维化功能。我们将分析 TGFB 的变化 肺中的信号传导和免疫细胞浸润可能是 Magp 保护免受感染的潜在机制 肺纤维化。最后，我们将采用我们最近开发的单核 RNASeq 方法以及 用于转座酶可及染色质 (snATAC)-Seq 的单核测定来表征成纤维细胞 表达 Magp1 和 2 的群体并评估全肺转录组和表观基因组如何变化 纤维化过程中 Magps 的缺失。该提案将为 Koenitzer 博士提供以下领域的指导培训： 所需的单细胞测序和生物信息学、成纤维细胞和 ECM 生物学以及肺组织学/成像 实现他作为一名医师科学家的独立目标。在专家科学顾问的监督下 委员会，他将实现职业里程碑，完成正式课程并发展持久的合作。 这项工作的结果将对 IPF 以外的其他间质性肺疾病产生影响，并使 Dr. Koenitzer 作为独立研究者开发治疗肺部疾病的新方法。

Project Summary/Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited treatment options and unclear pathogenesis. Extracellular matrix (ECM) deposition by proliferating fibroblast populations drives the progressive airflow limitation and hypoxia characteristic of IPF, mediated by the cytokine transforming growth factor beta (TGFB). Direct inhibition of TGFB leads to intolerable side effects, and there is increased interest in therapies that instead modulate its regulation. Microfibril-associated glycoproteins 1 and 2 (Magp1 and Magp2, or Magps) are fibroblast-produced proteins which are secreted to ECM, anchor to microfibrils, and bind TGFB to limit its signaling under basal conditions, but have no known role in fibrosis. Magp knockout (KO) mice exhibit increased TGFB signaling in various tissues and have immune cell alterations due to bone marrow TGFB effects. In a single cell RNASeq screen, the genes encoding these proteins were markedly upregulated in fibroblasts in fibrotic lungs, and combined KO of Magp1 and 2 led to more severe fibrosis after bleomycin injury in mice. This suggests that Magps act as inhibitors of fibrotic signaling and follow up studies in Magp1 and 2 individual KO mice show that deficiency of both proteins is required to produce a pro-fibrotic phenotype. Which fibroblasts populations produce Magps and how they limit fibrosis is unknown. We hypothesize that Magp expression is an adaptive response to lung injury and limits fibrosis by sequestering TGFB in ECM and will explore this central hypothesis through three specific aims: 1) Evaluate anti-fibrotic contributions of Magp1 and 2 in pulmonary fibrosis. 2) Evaluate Magp effects on TGFB signaling and immune responses in lung fibrosis. 3) Characterize Magp-producing fibroblast populations. Using combinations of KO and inducible/tissue-specific KO mice, we will determine the individual relationships of these proteins to fibrosis and whether they exhibit redundant antifibrotic functions in vivo. We will analyze alterations in TGFB signaling and immune cell infiltration in lung as possible underlying mechanisms of Magp protection from pulmonary fibrosis. Finally, we will employ our recently developed single nucleus RNASeq approach along with single nucleus assay for transposase-accessible chromatin (snATAC)-Seq to characterize the fibroblast populations that express Magp1 and 2 and evaluate how lung-wide transcriptomes and epigenomes change in the absence of Magps during fibrosis. This proposal will provide Dr. Koenitzer with the mentored training in single cell sequencing and bioinformatics, fibroblast and ECM biology, and lung histology/imaging needed to achieve his goal of independence as a physician-scientist. Under the oversight of an expert scientific advisory committee, He will realize career milestones, complete formal coursework, and develop lasting collaborations. Findings from this work will have implications beyond IPF in other interstitial lung diseases and enable Dr. Koenitzer to develop new approaches to the treatment of lung disease as an independent investigator.